This proposal addresses RFA-AI-10-003 Partnerships for Biodefense and brings together academic researchers, industry, MDR TB clinicians, BSL3 Labs, and field sites to develop a faster, quantitative, second line drug susceptibility test (DST) for multiple and extensively drug-resistant tuberculosis. The emergence of drug resistant TB, a Category C threat, has led the ominous situation of a highly transmissible and in some instances untreatable disease. Several Tb DST platforms are in use yet all have significant constraints, particularly turnaround time. We feel that we must persevere with a phenotypic culture based method, since direct molecular testing will remain poorly defined for second line drugs, but we have shown that can improve existing liquid methods with an earlier 3 day quantitative PCR-based readout that measures DNA amplification in the setting of drug. By using high-throughput small-volume microtiter plates, our method can yield both an accurate rapid qualitative result and a quantitative inhibition index akin to the MIC - a measurement that is clinically needed but rarely available for Tb. Because biosafety of MDR/XDR Tb for laboratory personnel is of utmost importance, we will then transform this assay into a novel-design, closed- system, disposable, qPCR-capable microfluidic chip. This microfluidic chamber can accurately meter the number of bacilli fed into each well, minimizing one area of poor reproducibility, and can also measure growth in situ by other parameters beyond qPCR, including a newly-invented DNA detection technique we term pinwheel. Thus the qPCR assay development and chip project will benefit from but are not tied to each other's success. The goal is an improved DST platform for second line drugs that yields faster information to better treat and decrease transmission of MDR/XDR Tb. NARRATIVE Drug susceptibility testing for MDR and XDR Tb is slow and fraught with technical difficulties. This proposal will develop a rapid, quantitative PCR-based diagnostic to detect susceptibility or resistance of Tb to second line drugs in a microplate format within 3 days. The assay will be adapted to a closed-system disposable chip to enhance biosafety for lab personnel and permit DNA quantification by our new 'pinwheel' approach. The results will be immediately usable by MDR Tb clinicians to better treat and decrease transmission of MDR/XDR Tb.